Image analysis of fluorescently labeled nuclear structures: DNA replication in focus

Abstract

In this thesis, we expanded the knowledge about some aspects of DNA replication in human cell nucleus with the help of microscopic techniques and advanced mathematical approaches. DNA replication was studied from several perspectives: Fluorescence detection of newborn DNA on the stretched DNA fibers, ultrastructural mappings of individual replication domains, fluorescence analysis of replication foci re-distribution in early S-phase and cold-dependent immunodetection of replication-coupled chromatin modulation. We started with measurements of average speed of a replication fork movement during S- phase. In agreement with previous studies on DNA spreads with radioactive labeling, the replication speed was two to three times slower in early S-phase than in late S-phase. Moreover, the exogenous dNTP supply accelerated the replication in the early S-phase while additional dNTPs had no effect on the speed of replication forks in late S-phase. Therefore the availability of dNTPs seems to be a rate-limiting factor for replication speed at the beginning of DNA synthesis. We then focused on in situ replication pattern at the EM level. Using an improved method for detection, replication domains, identified by clusters of many silver particles, were observed throughout the S-phase. While replication foci in early...